YAC transgenesis: a study of conditions to protect YAC DNA from breakage and a protocol for transfection.
Yeast artificial chromosomes (YACs) are providing a great boon to transgene technology by allowing the easy mutagenesis and study of very large DNAs. The large insert sizes of these vectors permit more accurate analysis of the regulation of transgene expression than smaller, more artificially assembled constructs. Transfection of mammalian cells by YACs can be accomplished by a number of methods; the most prevalent, using gel-purified DNA, is dependent upon compaction by salts to protect the large YAC DNA from breakage. We show that the common reliance on NaCl to compact YAC DNA sufficiently to protect it from breakage is not well-founded. Even the use of mixtures of polyamines and NaCl allows substantial damage to purified YACs. The use of polyamines alone in low salt buffers to compact YAC DNA provides the best protection from breakage and allows very effective transfection of murine embryonic stem (ES) cells. We provide a detailed method for ES cell transfection by YACs utilizing the DOTAP lipofection reagent that optimizes transfection efficiency and recovery of intact YACs.
Bauchwitz
R
R
Department of Genetics and Development, Columbia University, New York, NY 10032, USA. rpb@columbia.edu
Costantini
F
F
D000818:Animals; D001769:Blood; D018244:Chromosomes, Artificial, Yeast / Q000187:drug effects* / Q000235:genetics; D004249:DNA Damage / Q000187:drug effects; D004271:DNA, Fungal / Q000187:drug effects*; D005229:Fatty Acids, Monounsaturated; D005822:Genetic Vectors / Q000187:drug effects* / Q000235:genetics; D051379:Mice; D011073:Polyamines / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds; D012965:Sodium Chloride / Q000494:pharmacology; D013234:Stem Cells; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000187:drug effects* / Q000235:genetics
D004271:DNA, Fungal; D005229:Fatty Acids, Monounsaturated; D011073:Polyamines; D000644:Quaternary Ammonium Compounds; D012965:Sodium Chloride; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane